Cytokines comprise a variety of secreted proteins that regulate cell growth and differentiation. Cytokines like interleukin (IL)-2, IL-4 and IL-12, for example, dramatically influence the immune response, regulating both the magnitude and the character of the response. Understanding the molecular basis of this regulation should provide important insights that could shed light on disease pathogenesis and offer new therapeutic targets. In an effort to better understand cytokine signal transduction, we cloned a kinase, Jak3, which is a critical component for signaling by the subfamily of cytokines that share a common receptor subunit, the common gamma chain or gamma c (IL-2, IL-4, IL-7, IL-9 and IL-15). We further demonstrated that mutation of Jak3 is the basis for one form of primary immunodeficiency, autosomal recessive severe combined immunodeficiency (SCID). A major objective of this year was to improve our understanding of the structure and function of Jak3. In particular, we were interested in biochemically characterizing Jak3 mutants that resulted from missense mutations. One subset of patients of particular interest were those with mutations in the pseudokinase domain, a unique feature of Jak kinases. Through the analysis of these mutants, we have provided evidence that this domain has important regulatory functions and the pseudokinase and kinase domains interact. To better characterize how Jak3 is regulated and how it interacts with potential substrates, we also analyzed Jak3 autophosphorylation sites and have mutated all of the Jak3 tyrosine residues. We identified two important autophosphorylation sites (Y980 and Y981) which regulate Jak3 catalytic activity, positively and negatively, respectively. We have also identified several other authophosphorylation sites that we believe are involved in protein-protein interactions. We have also studied in detail how Jak3 and the common gamma chain associate. We have delimited a small region in the N-terminus that is primarily responsible for this interaction and have identified key residues involved in receptor binding. We have also begun investigating how Jak activation is translated to the activation of other pathways, e.g. the MAPK pathway. We demonstrated that the SH-2 containing phosphatase, SHP-2 is involved in IL-2 signaling and appears to serve important functions in linking IL-2 stimulation with activation of MAPK and STAT activation. Furthermore, we have identified a new IL-2 dependent tyrosine phosphorylated substrate, p98, that is associated with SHP-2, Grb2 and PI3 kinase. To better understand the regulation of expression of Jak3 we have also begun identifying and characterizing the Jak3 promoter. Finally, under a CRADA mechanism, we have used the information gained on Jak3, to begin to identify Jak3 antagonists which could be used therapeutically as a novel class of immunosuppressants. In contrast to IL-2 and other gamma c using cytokines, IL-12 is an essential cytokine that regulates cell mediated immunity and so-called Thelper1 (Th1) differentiation. We have previously determined that it activates the Janus kinases, Jak2 and Tyk2 and STAT4. Suprisingly, we also showed that interferon alpha (INF alpha) also activates STAT4, suggesting a role role for it in regulating cell-mediated immunity. Finally, we found that both interferon alpha and IL-12 induce a further modification of STAT4 namely, serine phosphorylation, indicating another commonality in interferon alpha and IL-12. We have performed mutational analysis of STAT4 and have identified a serine residue, which when mutated, dramatically inhibits STAT4 mediated transactivation. We are currently attempting to identify the kinase responsible for this phosphorylation event. To better understand the molecular basis of IL-12 action, a new objective was to begin defining IL-12-inducible genes. In view of the fact that INF alpha activates STAT4, we also sought to compare IL-12, IL-2, INF alpha and IL-4. Interestingly, we identified IRF-1 (interferon regulatory factor-1) as an IL-12 and IL-2 induced gene. We showed that this was not dependent upon production of IFN gamma or other factor. This is important as IRF-1 has been shown to be important in Th1 responses and IRF-1 -/- mice have defective IL-12 responses. We have also identified a number of other IL-12 inducible genes and have also identified unknown genes that are regulated by IL-12.